Effect of dietary zinc source, zinc concentration, and exogenous phytase on intestinal phytate degradation products, bone mineralization, and zinc status of broiler chickens

This study aimed to determine the effect of Zn source and dietary level on intestinal myo-inositol hexakisphosphate (InsP6) disappearance, intestinal accumulation of lower InsP and myo-inositol (MI), prececal mineral digestibility, bone mineralization, and Zn status of broilers without and with exogenous phytase in the feed. Male Ross 308 broilers were allocated in groups of 10 to 8 treatments with 8 pens each. Experimental diets were fed from d 7 to d 28 and contained 33 mg/kg dry matter plant-intrinsic Zn. Experimental factors were phytase supplementation (0 or 750 FTU/kg) and Zn source (none [0 mg/kg Zn], Zn-sulfate [30 mg/kg Zn], Zn-oxide [30 mg/kg Zn]). Additional treatments with 90 mg/kg Zn as Zn-sulfate or Zn-oxide and phytase were included to test the effect of Zn level. No Zn source or Zn level effects were observed for ADG, feed conversion ratio, prececal P digestibility, intestinal InsP6 disappearance, and bone ash concentration. However, those measurements were increased by exogenous phytase (P < 0.001), except the feed conversion ratio, which was decreased (P < 0.001). Ileal MI concentrations were affected by phytase × Zn source interaction (P < 0.030). Birds receiving exogenous phytase and Zn supplementation had the highest MI concentrations regardless of exogenous Zn source, whereas MI concentrations were intermediate for birds receiving exogenous phytase only. Exogenous phytase and exogenous Zn source increased the Zn concentration in bone and blood of broilers (P < 0.001). In conclusion, measures of exogenous phytase efficacy were not affected by phytase × Zn source interaction. Further studies are needed to rule out an effect from Zn sources other than those tested in this study and to investigate the effect of Zn supplementation on endogenous phosphatases. The missing effect of increasing Zn supplementation from 30 to 90 mg/kg in phytase-supplemented diets gives reason to reconsider the Zn supplementation level used by the industry.


INTRODUCTION
Zinc is an essential mineral in all living organisms, as it functions as a cofactor for many enzymes and as a second messenger (Kimura and Kambe, 2016;Brugger and Windisch, 2019).When the Zn supply is insufficient, growth and bone development can be impaired (Zeigler et al., 1961).The Zn supply recommendation for broiler chickens is 50 mg/kg DM according to the Gesellschaft f€ ur Ern€ ahrungsphysiologie (1999) or 40 mg/kg according to the National Research Council (1994).The intrinsic Zn concentration of common feedstuffs for poultry does not cover the Zn requirement, and the availability of intrinsic Zn is low due to dietary antagonists such as phytate (Maddiaiah et al., 1964).Hence, commercial broiler diets are usually supplemented with Zn, including high safety margins that are of concern in regard to environmental effects.
Phytate is the salt form of phytic acid (myo-inositol 1,2,3,4,5,6-hexakisphosphate [InsP 6 ]) and the primary storage form of P in plant-based feedstuffs.The availability of phytate-P is limited for nonruminant animals and different between animal species (Rodehutscord et al., 2022).Phytases (myo-inositol hexaphosphate phosphohydrolases) and other phosphatases are needed to induce cleavage of the phosphate groups from the myoinositol ring, making the phosphate absorbable.In order 1 to increase the digestibility of phytate-P, poultry diets are commonly supplemented with exogenous phytases whose main site of action is the proximal gastrointestinal tract.
Phytate can form insoluble complexes with (divalent) cations, such as Ca and Zn, especially at the pH prevailing in the small intestine (Oberleas et al., 1966;Champagne and Hinojosa, 1987).Calcium, for instance, has been shown to negatively impact InsP 6 disappearance in the small intestine of broilers at high dietary levels, especially in the presence of high dietary P levels (Sommerfeld et al., 2018b).As the stability of complexes between phytate and Zn was ranked the highest among all cations tested (Maenz et al., 1999), it is crucial to also consider Zn as a potential influencing factor of phytase efficacy.In vitro studies have shown an adverse effect of Zn on phytase efficacy.When Zn was added to a Naphytate solution, phosphate release by phytase was reduced, with the degree of reduction depending, among others, on the Zn source (Santos et al., 2015).It was hypothesized that the reduction might be due to the formation of insoluble Zn-phytate-complexes (Oberleas et al., 1966) rendering the InsP 6 unavailable for degradation by phytase.This hypothesis was supported by a study by Augspurger et al. (2004), in which tibia ash was significantly reduced in phytase supplemented diets when Zn was added at a pharmacologic level (800 mg/ kg).Besides the Zn level, the solubility of Zn sources could also affect the interaction with phytate, with faster dissolving Zn sources having greater potential for interaction than slower dissolving sources (Cardoso et al., 2021a).This was shown in vitro for 2 Cu sources differing in solubility, where phosphate release by phytase was lower when Cu was supplemented as Cu sulfate compared to Cu oxide (Hamdi et al., 2018).Because Znsulfate and Zn-oxide differ in their dissolution speed (Cardoso et al., 2021b), those 2 sources were chosen in the present study.To our knowledge, this hypothesis has never been tested in vivo for nonpharmacologic Zn levels in broilers.
Therefore, the first objective of this study was to investigate whether the supplementation of Zn, depending on the Zn source, affects phytase efficacy by determining intestinal InsP 6 disappearance, accumulation of lower InsP x , P digestibility, and bone mineralization.The second objective was to determine the Zn status, as determined by the Zn concentration of bone, blood, and liver of the birds, and whether it is affected by exogenous phytase, Zn source, and dietary Zn level.It was hypothesized that 1) Zn supplementation reduces phytase efficacy at a dose of 750 FTU/kg; and 2) phytase increases Zn concentration in bone, blood, and liver of birds.

Birds and Housing
The experiment was carried out at the Agricultural Experiment Station of the University of Hohenheim following the German Animal Welfare Legislation and approved by the Regierungspr€ asidium T€ ubingen (Approval no.HOH 65/21_460a).A total of 640 male Ross 308 broilers were obtained from a commercial hatchery (Br€ uterei S€ ud GmbH & Co. KG, Regenstauf, Germany) and distributed into 64 floor pens (115 £ 230 £ 260 cm) with wood shavings in groups of 10 birds.Each of the 8 dietary treatments was randomly assigned 8 pens in a randomized complete block design.From d 0 to d 7, birds received a pelleted nutrient-adequate corn−soybean meal-based pre-experimental starter diet according to Gesellschaft f€ ur Ern€ ahrungsphysiologie (1999).Birds were reallocated on d 7 to ensure even weight distribution across all treatments and blocks and received pelleted experimental grower diets from d 7 to d 28.Feed and tap water were provided ad libitum throughout the experiment.Birds were kept on perforated floors from d 16 to the end to prevent intake of marker (titanium dioxide) from the feces.For the first 3 d, the light schedule was 24L:0D; from d 3 until the end of the experiment, the light schedule was 18L:6D.The room temperature was continuously lowered from 34°C at the beginning to 22°C by the end of the experiment.Birds were checked twice daily to monitor the health status.

Experimental Diets
Diets were calculated to contain adequate levels of all nutrients according to Gesellschaft f€ ur Ern€ ahrungsphysiologie (1999), except non-phytate P and Zn.Diets were based on corn, soybean meal, rapeseed meal, and rice bran (Table 1).Titanium dioxide was used as an indigestible marker with an inclusion level of 5 g/kg.Eight experimental diets were prepared in a 2 £ 3 + 2 factorial arrangement of treatments.The factors Vitamin premix (GELAMIN, Germany), provided per kg of complete diet: 10,000 IU vitamin A, 1,000 IU vitamin D3, 2.4 mg vitamin K3, 0.1 mg biotin, 1 mg folic acid, 3 mg vitamin B1, 6 mg vitamin B2, 6 mg vitamin B6, 30 mg vitamin B12, 50 mg nicotinic acid, 14 mg pantothenic acid.
phytase level (0 (PHY−) and 750 FTU/kg (PHY+), Quantum Blue, AB Vista, Marlborough, United Kingdom (Escherichia coli derived 6-phytase)) and Zn source (none (Zn−) or 30 mg/kg of Zn sulfate heptahydrate (ZnSO 4 +), Sigma Aldrich, St. Louis, MO or 30 mg/kg of a modified Zn oxide (ZnO+), HiZox, Animine, Annecy, France) were included in the full-factorial design.The 2 other treatments were added to test the effect of Zn supplementation level (ZnSO 4 or ZnO at 90 mg/kg Zn [Zn++]) and included exogenous phytase (750 FTU/kg).The lower Zn supplementation level was chosen to meet the bird's requirement of 50 mg/kg DM (Gesellschaft f€ ur Ern€ ahrungsphysiologie, 1999).The higher Zn level was chosen on the basis of European regulations.For poultry, the Zn content in complete feed, including the intrinsic Zn content, must not exceed 120 mg/kg (DVO (EU) Nr. 2016/1095).The level of exogenous phytase (750 FTU/kg) was chosen in combination with the calculation of a high phytate-P level (3.5 g/kg DM) to ensure sufficient undegraded phytate is present in the small intestine to challenge interactions of phytate and Zn.Experimental diets were prepared by mixing all nonvariable ingredients (Table 1) to obtain a basal diet.The basal diet was then divided into 5 parts and supplemented with the corresponding quantity of ZnSO 4 (analyzed Zn concentration: 326 g/kg DM) or ZnO (analyzed Zn concentration: 731 g/kg DM) at the expense of corn.The mixtures with 90 mg/kg Zn were supplemented with the exogenous phytase product.The mixtures with 0 and 30 mg/kg Zn were split again and one half of each was supplemented with the exogenous phytase.After mixing, experimental diets were pelleted through a 3-mm pelleting matrix.The temperature of the pellets was recorded immediately after release from the press and did not exceed 78°C.Representative diet samples were taken and ground to pass a 0.5-mm sieve (Retsch ZM200, Retsch GmbH, Haan, Germany) or pulverized by a vibrating cup mill (PULVERISETTE 9, Fritsch GmbH, Idar-Oberstein, Germany).

Experimental Procedures
Birds and feed were weighed on d 7 and d 28 to calculate mortality-corrected average daily feed intake (ADFI), average daily gain (ADG), and feed conversion ratio (FCR) on a pen basis.In order to standardize the gut fill prior to sampling, the feed was withdrawn 2 h before slaughter and access to the feed was allowed again 1 h before slaughter.All birds from a pen were stunned with a gas mixture of 35% CO 2 , 35% N 2 , and 30% O 2 and one randomly selected bird was weighed individually, killed by decapitation, and trunk blood from this bird was collected in Na fluoride treated tubes.The plasma was obtained by immediate centrifugation at 2,000£g for 10 min.In order to obtain serum, the blood was collected in a tube without additive and tubes were centrifuged at 2,000£g for 10 min after resting at room temperature for 0.5 to 2 h.The liver was removed from the decapitated bird and weighed.All remaining broilers from the same pen were killed by CO 2 exposure.Digesta from the duodenum and jejunum combined (duo+jej) and the distal two thirds of the section between Meckel's diverticulum and 2 cm anterior to the ileo-ceco-colonic junction (herein defined as ileum) were collected by gentle squeezing and pooled on a pen basis.The left tibiotarsus (tibia) and left foot from 2 randomly selected birds per pen were collected.All samples were frozen immediately at À20°C, and digesta and liver were freeze-dried and stored at 4°C until further processing.Freeze-dried digesta were pulverized in a vibrating cup mill.

Chemical Analyses
Feed samples were analyzed for DM according to the official methods in Germany (Verband Deutscher Landwirtschaftlicher Untersuchungs-und Forschungsanstalten, 2007).Feed samples and digesta were analyzed for Ti according to the modified wet digestion method of Boguhn et al. (2009) followed by inductively coupled plasma optical emission spectrometry measurement, described by Zeller et al. (2015).For mineral analysis (Ca, P, Zn, Fe, Mn, and Cu), digesta and feed samples were digested using wet microwave digestion with nitric acid, followed by inductively coupled plasma optical emission spectrometry measurement (USEPA, 1994).
To determine InsP 3-6 isomers, the extraction and measurement in feed and digesta were conducted using the method of Zeller et al. (2015) with slight modifications described by Sommerfeld et al. (2018b).Using this methodology, it is not possible to separate the enantiomers.Because of co-elution, it was not possible to distinguish between isomers Ins(1,2,6)P 3 , Ins(1,4,5)P 3 , and Ins(2,4,5)P 3 .Hence, the term InsP 3x will be used for these isomers with unknown proportions.Myo-inositol (MI) in digesta and plasma were measured as described in Sommerfeld et al. (2018a).
Blood serum was analyzed for Zn, inorganic P (P i ), and Ca by IDEXX GmbH (IDEXX BioAnalytics, Kornwestheim, Germany).Zinc in blood serum was measured by optical emission spectrometry using Thermo-Fisher iCAP-Duo 7000.Calcium in blood serum was determined photometrically by color test in Beckman Olympus AU480.Inorganic P in blood serum was measured photometrically by UV-test in Beckman Olympus AU480.
Tibia was thawed and adhering soft tissue, cartilage caps, and fibula bones were removed manually.Feet were detached at the articulatio intertarsalis and used completely below that joint, including skin, claws, and tissues.Subsequently, tibiae and feet were rinsed with distilled water, carefully dabbed, and dried for 48 h (tibiae) and 72 h (feet) at 103°C in a convection oven (VL 115,VWR International GmbH,Darmstadt,Germany).After drying, bone samples were cooled in a desiccator and weighed.Ash content was determined at 600°C in a muffle furnace (L 40/11/B170, Nabertherm GmbH, Lilienthal, Germany) for 24 h (tibiae) and 48 h (feet) for individual tibiae and feet.Ashed tibiae and feet were weighed and pulverized.After wet microwave digestion with nitric acid, ground tibiae and feet ash samples were analyzed for Ca, P, and Zn using inductively coupled plasma optical emission spectrometry.The freeze-dried liver was pulverized and analyzed for Zn using the method described before for feed.
Phytase activity in feed samples was determined by AB Vista (Marlborough, United Kingdom) using the ELISA method and subsequent conversion of results to FTU per kilogram.
Difference between calculated and analyzed Zn concentrations did not exceed 5.5% (Table 2).Analyzed P concentrations were up to 11% higher than calculated, but the deviation between different treatments did not exceed 4%.The deviation of calculated and analyzed phytase activity was less than 10%.

Calculations and Statistical Analysis
Average daily gain, ADFI and FCR were calculated on a pen basis for the experimental phase (d 7−d 28) and corrected for mortality.
For performance, digesta, and bone, the pen was considered the experimental unit.The bird was considered the experimental unit for blood and liver since data were obtained from individual birds.
The InsP 6 disappearance and P and Ca digestibility were calculated for digesta of duo+jej and ileum using the following equation: Where yðXÞ is the disappearance or digestibility of X in % and X is the concentration of Ca, P, or InsP 6 in feed and digesta.Data were analyzed in a nested 2-factorial analysis of variance using the MIXED procedure of the software package SAS (version 9.4, SAS Institute Inc., Cary, NC).The following model was chosen: Where y ijkl is the response variable, m is the overall mean, a represents the fixed effect of ith treatment group (i = PHY+ ZnSO 4 ++ or PHY+ ZnO++ or others), b represents the fixed effect of jth exogenous phytase level within ith treatment group (j = 0 or 750 FTU/kg), g represents the fixed effect of kth Zn source within ith treatment group (k = Zn− or ZnSO 4 or ZnO), bg is the corresponding interaction effect of the jth exogenous phytase level and kth Zn source within ith treatment group, d is the random effect of lth block (l = 1 −8), and e ijkl is the residual error.The same model was fitted in a second version, where treatments were reparameterized as an 8-level treatment factor.The model was: with y ijkl = response variable, m = overall mean, t ijk = fixed effect of 8-level treatment factor, d l = random effect of block, and e ijkl = residual error.The latter was used to simplify the calculation of the following additional contrasts: 1. Zn level: 30 mg vs. 90 mg

Performance Traits
The average body weight of broilers at the start of the experimental phase (d 7) was 153 g.A total of 9 birds (1.4%) died during the experimental phase, but this was not related to treatments.Performance responses were not affected by PHY £ Zn source interaction.Phytase supplementation increased ADG by about 6 g/d (P < 0.001, Table 3) and reduced FCR by 0.12 (P < 0.001).Birds receiving ZnO+ had lower ADFI than birds receiving ZnSO 4 + or Zn− (P = 0.009).Both contrasts (Zn level, exogenous Zn source) were not significant for the performance traits measured.
InsP 6 Disappearance, P, Ca, and Zn Digestibility The InsP 6 disappearance, P digestibility and Ca digestibility in duo+jej and by the end of the ileum were unaffected by interaction (PHY £ Zn source), Zn source, or level, but were affected by exogenous phytase (Table 4).In PHY− treatments, InsP 6 disappearance was between 7% and 10.5% (duo+jej) or between 16.5% and 22.1% (ileum).Phytase supplementation increased InsP 6 disappearance to 51.4% to 54.3% in duo+jej and to 62.8% to 64.7% in the ileum (P < 0.001).Digestibility of P and Ca in duo+jej and ileum were increased and decreased, respectively, by phytase supplementation (P < 0.001, Table 4).Zinc digestibility in duo+jej and ileum was affected by PHY £ Zn source interaction (P < 0.010, Table 4).In PHY− treatments, Zn digestibility was at a similar level regardless of Zn source, whereas in PHY+ treatments, Zn digestibility was significantly higher in the Zn− treatment than in the ZnSO 4 + or ZnO+ treatment.In PHY+ treatments, birds receiving ZnSO 4 had lower Zn digestibility in duo +jej and ileum than birds receiving ZnO (P < 0.050).
Ileal concentrations of InsP 3-6 were not affected by interaction (PHY £ Zn source), Zn source, or level, but only by exogenous phytase (P < 0.001, Table 6). 2 Presented if the main effect was significant (P ≤ 0.050), and the 2-way interaction was not significant (P > 0.050).

Myo-Inositol
Myo-inositol concentration in duo+jej was increased in PHY+ treatments (Table 7; P < 0.001).Ileal MI concentration was affected by PHY £ Zn source interaction (P = 0.030).The lowest (P < 0.050) ileal MI concentration was observed in broilers fed PHY− treatments.The ileal MI concentration observed in birds fed PHY+ZnSO 4 + or PHY+ZnO+ was greater than in birds fed the PHY+Zn− treatment (P < 0.050).In plasma, the MI concentration was increased by phytase supplementation (P < 0.001) but was unaffected by Zn source.Birds fed 30 mg/kg Zn and phytase supplementation had higher plasma MI concentrations than birds fed 90 mg Zn and phytase supplementation (P = 0.013).

Blood Minerals and Liver
A significant interaction (PHY £ Zn source) was found for serum Zn concentrations (P = 0.014, Table 8).Lowest Zn concentrations were observed for the serum of broilers fed the PHY−Zn− treatment.Adding either an exogenous Zn source, phytase, or both, increased serum Zn concentrations (P < 0.001).Phytase supplementation increased serum P i concentrations and decreased serum Ca concentrations (P < 0.001).Liver Zn concentrations were higher for birds receiving Zn supplemented treatments independent of exogenous Zn source (P = 0.004).Phytase supplementation increased liver Zn concentration, total liver Zn quantity, and liver weight (P < 0.050).However, the phytase effect on liver weight disappeared when liver weights were corrected for the individual body weight of sampled animals (P > 0.050).

Bone Ash
Tibia and foot ash concentrations were increased by exogenous phytase (P < 0.001, Tables 9 and 10).Zinc concentrations in the tibia and foot ash were affected by interaction of PHY £ Zn source (P = 0.006).Both Zn and phytase supplementation significantly increased Zn concentrations in the tibia and foot ash, but the effect of Zn supplementation was greater than the effect of phytase supplementation.Birds receiving 30 mg Zn tended to have lower Zn concentrations in the tibia ash and foot ash than birds receiving 90 mg Zn (P < 0.100).Phytase supplementation decreased Ca concentrations in tibia ash (P = 0.003), and increased Ca concentrations in foot ash (P = 0.010).The concentration of P in tibia and foot ash was higher in PHY+ treatments compared to PHY− treatments (P < 0.001).

DISCUSSION
The first hypothesis that Zn supplementation reduces the efficacy of exogenous phytase at a dose of 750 FTU/kg could not be confirmed as the prececal InsP 6 disappearance, the prececal P digestibility and the bone ash concentrations were not affected by dietary Zn level or Zn source (solubility).In contrast, the second 2 Presented if the main effect was significant (P ≤ 0.050), and the 2-way interaction was not significant (P > 0.050).
hypothesis, that phytase supplementation increases the Zn concentrations in bone, blood, and liver was confirmed.

Effects of Zn Source and Level on Phytase Efficacy
The supplementation of phytase significantly increased the InsP 6 disappearance in duo+jej and by the end of the ileum, and the concentrations of lower InsP x in duo+jej and ileum, confirming results from previous studies (Zeller et al., 2015;Sommerfeld et al., 2018b;K€ unzel et al., 2021).An interaction of phytase and Zn source was only observed in duo+jej for Ins(1,2,3,4)P 4 and Ins(1,2,3,4,5)P 5 .The concentration of both isomers was not affected by the Zn source in PHY− treatments but in PHY+ treatments.However, this Zn source effect on some lower InsP x was not significant anymore by the end of the ileum.The absence of interaction (PHY £ Zn source) and the non-significant contrast for 30 mg vs. 90 mg Zn supplementation for InsP 6 disappearance and accumulation of lower InsP x by the end of the ileum showed that neither Zn source nor Zn level affected phytase efficacy on a level that is relevant for the bird.Of note, the present study used an InsP 6 -P concentration in the feed of 3.3 g/kg DM, which was higher than in common diets based on soybean meal, corn, and other cereal grains.This was intended to provide a high amount of substrate for Zn to bind to and the exogenous enzyme to hydrolyze, thus creating a greater potential for interactions in the digestive tract.
Changes in phytase efficacy could occur through interactions between Zn and phytate.The results of the present study confirm the hypothesis of Schlegel et al. (2010) that supplemented Zn, independent of the source (chelates or sulfates), does not interact with phytate, at least not to the extent that it would influence InsP 6 degradation.However, this finding contradicts in vitro studies that reported a negative effect of Zn on phytase efficacy (Maenz et al., 1999), which depends on Zn level and source (Santos et al., 2015).Differences in the results of in vitro studies compared to the present study might be attributed to the different sources of InsP 6 , which can impact phytase efficacy and susceptibility to interaction with minerals (Sommerfeld and Santos, 2021).In vitro studies often use Na phytate (Sommerfeld and Santos, 2021), whereas in the present study, InsP 6 originated from corn, soybean meal, rapeseed meal, and rice bran.Also, different phytase origins (e.g., E. coli or Aspergillus niger) might cause contradictory results, as they seem to have different sensitivity to Zn supplementation (Santos et al., 2015).Another difference between in vitro studies (Santos et al., 2015) and the present in vivo study is the order in which the reactions or interactions between Zn and phytate or exogenous phytase and phytate occur.In vitro, all interactions occur simultaneously, while in vivo, different conditions along the digestive tract cause potential interactions to occur in different sections to a different degree.Further, the lack of interaction of PHY £ Zn source contradicts an in vivo study, where adverse effects of Zn supplementation on tibia ash were found in the presence of exogenous phytase (Augspurger et al., 2004).The conflicting results might be attributed to the different Zn levels.In the study by Augspurger et al. (2004), the Zn level was much higher (800 mg/kg, Zn from Zn chloride) than the highest Zn level used in the present study.Zinc  5 Presented if the main effect was significant (P ≤ 0.050), and the 2-way interaction was not significant (P > 0.050).
digestibility data on an intestinal level could not provide further insight into interactions of Zn and phytate.
Because of endogenous Zn losses that depend on the level of Zn supply (Brugger and Windisch, 2019), the value of Zn digestibility data to study Zn interactions is limited.
Myo-inositol is obtained by complete dephosphorylation of InsP 6 in the gastrointestinal tract.To our knowledge, this is the first study looking at intestinal MI concentrations concerning dietary Zn level and Zn source in broiler chickens.As observed in other studies, a consistent increase of MI concentration was found in  4 Presented if the main effect was significant (P ≤ 0.050), and the 2-way interaction was not significant (P > 0.050).
Table 7.Effect of phytase supplementation (PHY), Zn source, and dietary level on myo-inositol concentration in duodenum and jejunum (duo + jej), ileum and plasma of broiler chickens fed the experimental diets from d 7 to d 28.Means within a column not showing a common superscript differ (P ≤ 0.050).

Duo+jej
1 Main effect estimates represent treatments within the full-factorial design only; treatments with Zn level of 90 mg/kg are not included.
2 Presented if the main effect was significant (P ≤ 0.050), and the 2-way interaction was not significant (P > 0.050).duo+jej, ileum, and plasma due to phytase supplementation (Beeson et al., 2017;Sommerfeld et al., 2018a,b;Novotny et al., 2023).The supplementation of Zn, in addition to exogenous phytase, resulted in increased ileal MI concentrations compared to phytase supplementation alone.The lower ileal MI concentration in the PHY+ Zn− treatment is consistent with the numerically higher ileal concentration of the isomers Ins(1,2,5,6)P 4 of that treatment compared to the PHY+ ZnSO 4 + and PHY+ ZnO+ treatments, indicating a decelerated degradation and leading to lower ileal MI concentrations in the PHY+ Zn− treatment.As Zn is a cofactor for alkaline phosphatase (McCall et al., 2000), Zn supplementation might increase endogenous alkaline phosphatase activity (Williams, 1972).It can be assumed that 2 different "types" of interaction can affect the activity of alkaline phosphatases.On the one hand, in treatments without Zn supplementation, an internal interaction (triggered by Zn deficiency) could influence the activity of the phosphatases.On the other hand, an external interaction between Zn and phytate/phytase could influence the activity.Endogenous phosphatases are produced by either enterocytes or microorganisms and contribute to the degradation of InsP, especially of lower InsP (Huber, 2016;Rodehutscord and Rosenfelder, 2016).However, no differences in the ileal MI concentrations were observed due to Zn in PHY− treatments.The lack of an effect of Zn in PHY− treatments could be due to a lower mucosal phosphatase activity due to the reduced intestinal flow of lower InsPs in PHY− treatments compared with PHY+ treatments (Novotny et al., 2023).The smaller scope for Zn to affect the activity of phosphatases in PHY− treatments might have caused that the effect of Zn was not visible.
As expected, phytase supplementation significantly increased P digestibility in duo+jej and by the end of the ileum, and bone ash.Although it has been suggested for piglets that Ca 2+ and Zn 2+ compete for a non-specific transporter in the brush border membrane (Bertolo et al., 2001), in the present study, no effects of Zn source or dietary Zn level were found on Ca digestibility.However, phytase supplementation significantly decreased Ca digestibility in duo+jej and by the end of the ileum.Previously published studies on this topic show divergent results.Some studies observed an increase in prececal Ca digestibility due to phytase supplementation (Sommerfeld et al., 2018a;Krieg et al., 2021;Li et al., 2021), while others found no effect (Sommerfeld et al., 2018b;Krieg et al., 2021) or a decreased prececal Ca digestibility (Olukosi et al., 2013;Krieg et al., 2021).In contrast to P, where excretion with urine is low, Ca excretion with urine is variable and can be markedly high.Thus, complementary collection of total excreta is needed for a comprehensive evaluation of effects on Ca metabolism.
As shown in several other studies (K€ unzel et al., 2019;Babatunde et al., 2020;K€ unzel et al., 2021), tibia and foot ash quantity (g/bone), as well as tibia and foot ash concentration were significantly increased by phytase supplementation.Consistent with that, P and Ca 3 Presented if the main effect was significant (P ≤ 0.050), and the 2-way interaction was not significant (P > 0.050).
quantities and P concentration in tibia and foot ash were significantly increased with phytase supplementation.However, Ca concentration in tibia ash was significantly reduced due to phytase supplementation.The response to phytase supplementation in terms of P concentrations showed similar pattern in foot ash and blood serum (coefficient of correlation (r) = 0.73, P < 0.001).Phytase supplementation increased P i serum concentration and decreased Ca serum concentration, which has also been observed in other studies (Sebastian et al., 1996;K€ unzel et al., 2019).The homeostasis of Ca and P is interdependent.Low P supply of birds receiving PHY− treatments might have caused a reduced incorporation of P and Ca into the bones to maintain the required blood P level, leading to elevated blood Ca levels (Proszkowiec-Weglarz and Angel, 2013).The present study confirmed results from Schlegel et al. (2010) and Olukosi et al. (2018) that tibia ash concentrations were not affected by dietary Zn level or Zn source.In contrast, Ao et al. (2007) found an increase in tibia ash (mg/tibia) due to the supplementation of a chelated Zn source, and Augspurger et al. (2004) observed a decrease in tibia ash concentration and quantity in treatments supplemented with phytase and high levels of Zn chloride (800 mg/kg Zn).As mentioned before, differences between the latter and the present study might be partly explained by the different Zn supplementation levels.Another factor might be the exogenous Zn source used.The 2 exogenous Zn sources used in the present study showed no difference in their effect on phytase efficacy or related measures.However, a difference between other Zn sources than ZnO (HiZox) and ZnSO 4 cannot be ruled out.

Effects of Phytase Supplementation on Zn Traits
All Zn traits investigated were highly correlated with one another, particularly bone and serum Zn concentrations (Zn tibia ash and Zn foot ash concentration: r = 0.98; Zn serum and Zn tibia ash concentration: r = 0.78; (P < 0.001)).As observed in other studies, phytase supplementation increased Zn concentrations in serum and tibia ash (Ao et al., 2007;Schlegel et al., 2010).This increase can be attributed to the increased degradation of InsP 6 in the gastrointestinal tract and the associated release of Zn (Yi et al., 1996;Mohanna and Nys, 1999a).In the present study, 30 mg/kg Zn supplementation further increased Zn concentrations in serum and tibia ash in PHY+ treatments, whereby the effect was independent of the exogenous Zn source used.A supplementation of 90 mg/kg Zn did not further increase Zn concentrations in serum and tibia ash.No difference due to the exogenous Zn source (15 mg/kg Zn as ZnSO 4 vs.Zn glycine chelate) was also observed in the study by Schlegel et al. (2010).However, unlike the present study, combining phytase and Zn 3 Presented if the main effect was significant (P ≤ 0.050), and the 2-way interaction was not significant (P > 0.050).
supplementation did not further increase Zn concentrations in plasma.Previous studies have reported that blood and tibia Zn concentrations plateau at a certain level of Zn supplementation (Wedekind et al., 1992;Mohanna and Nys, 1999b).The higher dietary phytate-P concentration and the lower dietary intrinsic Zn concentration in the present study possibly caused the blood Zn concentration to plateau only when Zn and phytase were supplemented together.In contrast, in the study of Schlegel et al. (2010) a plateau of blood Zn concentration was probably already reached with phytase supplementation alone, whereas the tibia ash Zn concentration did not reach a plateau with phytase supplementation alone.As in the present study, Ao et al. (2007) observed increased liver Zn concentrations due to Zn supplementation.However, contrary to the present study, phytase supplementation in their study did not significantly affect liver Zn concentration.The difference might be partly explained by the different phytase supplementation levels (750 FTU/kg vs. 500 FTU/kg) and dietary phytate-P concentrations.In the present study, no differences in liver Zn concentrations were found depending on the exogenous Zn source used.Consistent with this, no differences in liver Zn concentrations were observed by Azad et al. (2017) when comparing ZnSO 4 with Zn methionine and Zn-enriched yeast.
With respect to the Zn status, growth performance appears not to be a sensitive trait in the present study.The ADG was not affected by Zn source or supplementation level indicating that intrinsic Zn was sufficient for growth when phytase was added.This is consistent with the results from studies by Schlegel et al. (2010) and Burrell et al. (2004), whereas in other studies the intrinsic Zn concentration was not sufficient for growth (Yi et al., 1996;Ao et al., 2007).Compared to the present study, intrinsic Zn concentrations were lower in the studies where Zn was limiting for growth (30 mg/kg vs. 20 mg/kg, 25 mg/kg).As mentioned before, the diet composition affects the availability of intrinsic Zn, which makes it difficult to determine the optimal Zn content in broiler diets.

CONCLUSION AND IMPLICATIONS
In conclusion, Zn supplementation at nonpharmacologic levels did not reduce exogenous phytase efficacy.Previously reported effects of Zn supplementation on phytase efficacy could be due to very high Zn supplementation levels or differences in the solubility of InsP 6 sources.Further studies are required to rule out an effect on exogenous phytase efficacy from Zn sources other than those tested in this study.Also, further research is needed on the activity of endogenous phosphatases, as the effect of Zn supplementation on these enzymes requires further investigation.It was confirmed that phytase supplementation increases the Zn concentrations in bone, blood serum, and liver.Since in the presence of phytase, supplementation of 90 mg/kg Zn did 3 Presented if the main effect was significant (P ≤ 0.050), and the 2-way interaction was not significant (P > 0.050).
not confer any benefit in comparison to 30 mg/kg Zn, the level of Zn supplementation in phytase-supplemented broiler diets used by the industry may be reconsidered.

Table 1 .
Ingredient composition of the basal diets.
1Mineral premix (BASU Mineralfutter GmbH, Germany) provided per kg of complete diet: 45 mg Ca from calcium carbonate, 74 mg Mn from manganese sulfate, 23 mg Fe from iron sulfate, 7 mg Cu from copper sulfate, 0.6 mg I from calcium iodate, 0.2 mg Se from sodium selenite.2

Table 2 .
Calculated and analyzed composition of the experimental diets.

Table 3 .
Effect of phytase supplementation, Zn source, and dietary level on performance traits in broiler chickens fed the experimental diets from d 7 to d 28.
Abbreviations: ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion ratio; SEM, standard error of the mean.Data are given as least square means; n = 8 pens.Means within a column not showing a common superscript differ (P ≤ 0.050). 1 Main effect estimates represent treatments within the full-factorial design only; treatments with Zn level of 90 mg/kg are not included.

Table 4 .
Effect of phytase supplementation (PHY), Zn source and dietary level on Ca, P, and Zn digestibility and InsP 6 disappearance in duodenum and jejunum (duo + jej) and ileum in broiler chickens fed the experimental diets from d 7 to d 28.
SEM, standard error of the mean.Data are given as least square means; n = 8 pens.Means within a column not showing a common superscript differ (P ≤ 0.050). 1 Main effect estimates represent treatments within the full-factorial design only; treatments with Zn level of 90 mg/kg are not included.

Table 5 .
Effect of phytase supplementation (PHY), Zn source and dietary level on InsP x concentrations (mmol/g DM) in duodenum and jejunum (duo + jej) in broiler chickens fed the experimental diets from d 7 to d 28.

Table 6 .
Effect of phytase supplementation (PHY), Zn source, and dietary level on InsP x concentrations (mmol/g DM) in ileum in broiler chickens fed the experimental diets from d 7 to d 28.not quantifiable in majority of samples, limit of quantification of Ins(1,2,3,4,6)P 5 and Ins(1,2,5,6)P 4 : 0.3 mmol/g.3Main effect estimates represent treatments within the full-factorial design only; treatments with Zn level of 90 mg/kg are not included.

Table 8 .
Effect of phytase supplementation (PHY), Zn source, and dietary level on blood minerals and liver characteristics in broiler chickens fed the experimental diets from d 7 to d 28.Main effect estimates represent treatments within the full-factorial design only; treatments with Zn level of 90 mg/kg are not included.
1Liver weight corrected for individual body weight. 2

Table 9 .
Effect of phytase supplementation (PHY), Zn source, and dietary level on tibia mineralization in broiler chickens fed the experimental diets from d 7 to d 28.
SEM, standard error of the mean.Data are given as least square means; n = 8 pens.Means within a column not showing a common superscript differ (P ≤ 0.050). 1 Dry matter basis. 2 Main effect estimates represent treatments within the full-factorial design only; treatments with Zn level of 90 mg/kg are not included.

Table 10 .
Effect of phytase supplementation (PHY), Zn source, and dietary level on foot mineralization in broiler chickens fed the experimental diet from d 7 to d 28.error of the mean.Data are given as least square means; n = 8 pens.Means within a column not showing a common superscript differ (P ≤ 0.050).
1 Dry matter basis. 2 Main effect estimates represent treatments within the full-factorial design only; treatments with Zn level of 90 mg/kg are not included.